|
| Status |
Public on Jun 18, 2014 |
| Title |
ATM-RP105-Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Splenic IgM+ B cell
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129sv
tissue: Spleen
target locus: c-myc intron one
genotype: ATM-/-
treatment: RP105
|
| Treatment protocol |
Retroviral infection with I-SceI-GFP-expressing retrovirus was performed after 24 hrs following α-CD40/IL4 stimulation and cells harvested at day 4. For RP105 stimulation, infection was performed after 48hrs and cells harvested at day 5. For indicated experiments, ATM inhibitor KU55933 (10μM, Tocris) was added at time of infection and maintained for the whole duration of the stimulation.
|
| Growth protocol |
Splenic B cells were isolated from mice at an age of 6-8 weeks and cultured in 15% FBS-containing RPMI medium supplemented with α-CD40 (1 μg/mL) plus IL4 (20 ng/mL) or RP105 (2 μg/mL).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were collected and treated withlysis buffer (200mM NaCl, 0.2% SDS, 100mM Tris-HCl pH7.5, 5mM EDTA pH8.0, 200ug/mL proteinase K) at 55degC overnight before precipitation with isopropanol
Genomic DNA was digested with HaeIII enzyme, A-tailing on 3' end (Klenow exo-), and adapter ligation. All libraries were blocked by EcoRV and XbaI to suppress amplification of germline or perfect repaired breaksites before PCR amplification with biotinylated primers, then streptavidin bead enrichment, emulsion PCR with nested primers, and a third PCR to include 454 or Miseq specific primers for sequencing.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
25xI-SceI cassette integrated in one allele of c-myc intron one
HTGTS
|
| Data processing |
Standard basecalling formats for 454 and Miseq reads
Miseq paired end reads required trimming and stitching using ea-utilsv1.1.2 (http://code.google.com/p/ea-utils) followed by CD-HITv4.5 (Li & Godzik, Bioinformatics 2006) to remove 100% duplicates
Both 454 and Miseq reads are aligned using Blat (Kent, Genome Research 2002) with a masked targeted locus. Reads containing artifacts or duplicate junctions were filtered out.
Genome_build: mm9
Supplementary_files_format_and_content: tab delimited text files contain filtered unique junctions and include the following information: sequence ID (Qname); beginning (Qstart) and end (Qend) nucleotide position within the read of alignment; orientation of junction (strand); chromosome (Tname) and position (Tstart and Tend) of alignment to the genome build; the entire read sequence (raw) including stitched paired end reads; the experiment assocaited with the read (ExpID); and the RefGene feature the alignment is associated with (Genes; NA=no annotation)
|
| |
|
| Submission date |
Jun 17, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Jiazhi Hu |
| E-mail(s) |
heartlead@gmail.com
|
| Organization name |
Children's Hospital Boston
|
| Department |
PCMM
|
| Lab |
Frederick Alt
|
| Street address |
300 Longwood Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (1) |
| GSE58599 |
High-throughput sequencing of genome-wide translocations from stimulated wild-type or ATM-deficient B cells |
|
| Relations |
| BioSample |
SAMN02864987 |
| SRA |
SRX609852 |
